2960 exam 3

HP1 maintained constitutive heterochromatin. (always off) - The fly eye is an experimental system to study constitutive heterochromatin.

The white gene is named for its mutant phenotype When the white gene is next to heterochromatin, occasionally its expression is silenced. A histone methylation "reader", HP1, helps heterochromatin spread by recruiting HMT A heterochromatin boundary is maintained by competing activating histone modifiers (HATs and kinases).

X-chromosome inactivation

Xist RNA

III.Intron splicing (removal of intron sequences) B. mRNA splicing 4. Exons (molecular definition

a. Branch point and 3' splice site which bind to U2 snRNP and Protein Complex (PC), respectively b. 5' splice site which binds to U1 snRNP are essential

polysome

multiple ribosomes translating a single mRNA transcript

DNA sequence that determines the site of transcription initiation

promoter, -10,-35

TFIIB

start site selection; stabilize TBP-TATA complex; pol II/TFIIF recruitment recognition factor recognizes BRE element in promoters; accurately positions RNA polymerase at the start site of transcription Binds to TBP; recruits Pol II-TFIIF complex

CRISPR/Cas9

• A natural mechanism used by bacteria as a defense mechanism against virus infection

Comparing mouse model results to human data

• The microbiota of obese human subjects harbored fewer Bacteroidetes than lean subjects • After being placed on fat-restricted or carbohydrate-restricted diets the microbiota of the obese subjects changed over time

Is obesity associated with an alteration in the gut microbiome?

• This can be addressed using a mouse model for obesity. • Remember the OB gene production leptin. • Ley et al analyzed 16SrRNA sequence from microbes in intestine of ob and lean mice (fed same diet): • Focused on 2 phyla: Firmicutes & Bacteroidetes

histone modifications - "Writers"

"Writers" covalently modify histone amino acids - methyl or acetyl transferases

Phosphorylation of the C-terminal domain (CTD) of RNA Polymerase II helps ___

"open" the transcription bubble Pre-Initiation Complex- DNA is not yet "opened" to expose template strand for transcription initiation

Activators have two separable domains for:

(1) DNA binding and (2) activation If DNA binding domains are "swapped out" for one another, activation still works! In other words, these domains which are interchangeable.

Overview of Transcription

*RNA Polymerase* binds to DNA template: at promoter • DNA helix unwound to form transcription bubble. Rewound after transcribed • One strand is the *template*: for RNA synthesis • For any one gene, only one strand is the template • *RNA* is synthesized from rNTPs, 5' to 3' direction • Form base pairs, complementary to the bases on *DNA template* • 5' end of *transcript* is displaced from template as polymerase moves

Nucleotide excision repair

*Recognizes* bulky distortion of helix; *Removes* fragment containing the damage and *Replaces* it by new synthesis of DNA In bacteria, a UvrAB complex *recognizes* damage (distortion) and recruits UvrB. UvrB and UvrC cut damaged strand 5 and 3 to damage site. UvrD , a helicase, unwinds the DNA and *removes* the damaged oligonucleotide. DNA Polymerase 1 *replaces* the missing DNA.

Question: If tRNA molecules carrying the correct amino acids are the key to reading the genetic code... The critical question then becomes: How are specific amino acids attached to specific tRNA molecules?

- Amino-acyl tRNA - synthetases • Enzymes responsible for linking specific amino acids to the tRNAs that recognize the codons for that specific amino acid

Pol II

- TRANSCRIPTION INITIATION, ELONGATION, TERMINATION - recruitment of mRNA capping enzymes - transcription- coupled recruitment of splicing and 3' end processing factors -CTD phosphorylation, glycosylation, and ubiquitination The RNA polymerase complex responsible for transcription of protein-coding genes.

Amino-acyl tRNA synthetases charge tRNAs with specific amino acids 2 step

- activate amino acid - transfer to tRNA

TFIID

- core promoter binding factor - coactivator - protein kinase - ubiquitin - activating/ conjugating activity - histone acetltransferase the first general transcription factor to bind the promoter, binds to the TATA box through the TATA binding protein (TBP) causes a distortion in the DNA helix allowing the recruitment of other transcription factors

Levels of Chromatin Packing

- dna double helix - nucleosomes - chromatin - scaffold associated chromatin - condensed heterochromatin - compacted chromosome

The sigma(s) subunit of E. coli RNA polymerase binds to the

-10 and -35 consensus sequence elements (promoter)

Specific binding sites recognized by RNA polymerase sigma factor

-10, -35

TFIIH

-ATPase activity for transcription initiation and promoter clearance - helicase activity for promoter opening -transcription - coupled nucleotide excision repair - kinase activity for phosphorylating polII CTD -E3 ubiquitin ligase activity contains a DNA helicase to unwind DNA and activates RNA polymerase by phosphorylation unwinds DNA at the transcription start point, phosphorylates Ser5 of the RNA polymerase CTD; releases RNA polymerase from the promoter

How Does Gal4 Activate Transcription in Yeast?

-galactose: Gal4 is inhibited by Gal80 bound to activation domain +galactose: Galactose/Gal3 binds and sequesters Gal80 in the cytoplasm. Gal4 activation domain binds a histone actetylase coactivator complex (called SAGA) and mediator, which recruit GTFs and PolII to promoter.

Translation in Prokaryotes

-transcription and translation are simultaneous in bacteria -DNA is in cytoplasm -no mRNA editing -ribosomes read mRNa as it is being transcribed

In describing proteins that interact with the amino acids of the histone tails, we discussed the classifications writers, erasers and readers. For each, describe in one sentence what those classifications mean, and give an example of each type of modifier.

-writers covalently add a chemical group to histone tails. Examples include (only one is required): HAT (aka histone acetylase or histone acetyltransferase), HMT (aka histone methylase or histone methyltransferase) -erasers remove a chemical group from histone tails. Examples include (only one is required): HDAC (aka histone deacetylase) or histone demethylase -readers bind to a chemical group on histone tails. Examples include (only one is required): HP1...

Cis and trans-acting components

1 copy in genome 1 copy on F' plasmid • *lacI Repressor* encoded by one locus can function at second locus: • acts "in trans" • Can diffuse, bind to other *lacO* sites in the cell • *lacO* a DNA element • Regulates expression of gene directly adjacent • *lacO* acts "in cis"

TFIIH has two roles during transcription initiation

1) DNA "melting" catalyzed by a TFIIH helicase. 2) Phosphorylation of the carboxy terminal domain (CTD) of RNA pol II by a TFIIH kinase.

Three phases of transcription

1) Initiation: RNA polymerase "recognizes" and binds to promoter sequences, determines starting point for transcription (+1) 2) Elongation: RNA polymerase monitors binding of rNTP to next base on template. If match, catalyzes bond formation 3) Termination: RNA Polymerase pauses and dissociates from the template. Usually at designated termination site

Translation is a very accurate process. Which steps in translation include a process that helps assure the accuracy of the translation process? Explain your answer.

1)Establishing the 30S initiation complex at the first AUG of an open reading frame (ORF). The spacing between the Shine-Dalgarno sequence (aka Ribsome Binding Site or RBS) and the complementary sequence on the 16S rRNA in the 30S ribosomone ensures that the correct start codon is placed in the P site, and thus the correct ORF is translated. 2)Linking of the correct amino acid to the appropriate tRNA, to generatea "charged tRNA". This is carried out by aminoacyl-tRNA synthestases, which have a mechanism that helps ensure that the correct amino acidis linked to a tRNA with the corresponding anticodon sequence. The"mischarging" rate is low: 1/105-1/104 . 3)Insertion of charged tRNA into the A siteof the ribosome. Correct interactions between the tRNA anticodon loop and the codon sequences must occur before the hydrolysis of GTP on EF-Tuand the release of EF-Tu from the charged tRNA.

MBD proteins recognize methylated DNA and help maintain chromatin in an off state 3 steps

1. 5MethylC Binding Domain (MBD) proteins recognize 5meC modified DNA. 2. Then, MBD proteins recruit histone deacetylases (HDACs). 3. De-acetylated chromatin is in an off state.

snRNA-mediated splicing

1. After U1 and U2 have bound to the 5' splice site and branch point, respectively, U5 binds to bring them together. 2. U4 and U6 binds to the spliceosome. Subsequently, U4 leaves and releases U6. 3. U6 base pairs to U2. 4. U6 displaces U1 at the 5' SS. 5. Exons clipped and ligated and the lariat (intron) released.

Double-strand break repair comes in two flavors:

1. Direct rejoining of broken ends-called *non- homologous end-joining (NHEJ)*. Confined mostly to to eukaryotic cells. 2. Copying of broken sequence from sister chromatid in a cell that has a second good copy of the broken sequence following DNA replication. Called *homologous recombination*.

There are several types of pathways for repairing damaged DNA

1. Direct reversal of DNA damage Example, in E. coli *photo-reactivated* repair of pyrimidine dimers caused by UV light 2. Excision repair pathways Base excision repair Nucleotide excision repair 3. Error-prone repair pathways Damage is not removed but copied 4. Strand-break repair Double-strand break repair

How to study microbes

1. Examine samples under microscope 1. Culture(when possible) • A large number of microbes seen under a microscope cannot currently be grown under laboratory conditions 2. Culture-independent techniques • Isolate total DNA from environmental sample & sequence

Targets of activation domains are recruited to the promoter by protein-protein interactions; among these targets, which are called co-activators, are: 3

1. General transcription factors a. TFIID-major one (TBP with TAFs) b. Mediator complex-associated with RNAP 2. Histone Code Writers (Most important are HATs, but also certain Histone Methyltransferases) 3. Chromatin remodeling complexes

Functions of the 5' Cap:

1. Increase transcript stability by protecting against ribonucleases. 2. Increase translation efficiency; 5' cap binds to translation initiation factors.

Acetylation of lysines in histone tails probably does two things:

1. It loosens chromatin structure by relieving nucleosome- nucleosome interactions and histone-DNA interactions. 2. It provides binding sites for proteins that facilitate activation. (Readers of the histone code.)

5' mRNA capping

1. Mechanism of 5 Removal of the terminal Pi from the 5' end. 2. A guanine cap it attached to the 5' end using GTP (releasing PPi). This is a 5'-to-5' linkage. 3. Methylation of guanine and some ribose

Genetic approaches for investigating gene function 3 steps of Reverse genetics & Genome editing

1. Reducing expression of a gene - RNAi 2. Introducing imprecise changes in a gene: - insertions or deletions - can result in loss of function 3. Introducing precise changes: eg. specific nucleotide (homology Directed Repair = HDR) - CRISPR/Cas9

Features of Transcription in Eukaryotes 3

1. Spatial separation of transcription and translation in eukaryotes 2. More complex transcriptional regulation in eukaryotes: •Three RNA polymerases •General Transcription Factors (GTFs) •cis-acting elements (DNA regulatory sequences that bind proteins) are more varied and can be positioned in different configurations relative to the coding sequence 3. Extensive processing of primary transcript destined to become mRNA in eukaryotes

Sources of mutations

1. Spontaneousmutations Examples: errors during replication, spontaneous lesions, repeat expansion (which is really a replication error) 2. Induced mutations (used in the lab to create mutations) Examples: chemicals like base analogs, intercalating agents, UV light, X-rays

the steps of pre-initiation complex assembly DeBoRaH

1. TFIID binds to core promoter elements 2. TFIIB binds to TFIID/DNA 3. Pol2 RNA Polymerase binds to TFIIB/TFIID/DNA 4. TFIIH binds to complex 5. TFIIH opens DNA and phosphorylates CTD to allow initiation

Assembly of the Spliceosome

1. U1 snRNA in the U1 snRNP binds to the 5' splice site. 2. The protein complex binds to the 3' splice site which recruits U2 snRNP to the branch point. 3. U4, U5, and U6 complex binds. 4. U1 and U4 are removed, leaving only U2, U5 and U6 .

Mediator:

1. is required for transcription of of most genes in cells; i.e. in a chromatin context! 2. interacts with RNA Polymerase, other GTFs, and with activator proteins bound to far-away enhancers.

The Lac operon: Cis & Trans-acting factors Why are the results of adding a plasmid copy different for lacI- and lacP-?

1. lacI expressed from plasmid can still bind lacO. •lacI works in *trans* 2. lacP sequence on plasmid can bind RNA polymerase, but it can only initiate transcription for the lacZY if genes directly adjacent. It can not restore expression of lac genes on chromosome. •lac P works in *cis*

Sequential recruitment of *general transcription factors* (GTFs) & assembly of the eukaryotic RNA pol II Pre-Initiation Complex (PIC)

1st: TATA-box Binding Protein (TBP) as part of TFIID; the anchor 2nd: TFIIB 3rd: RNA Pol II (binds to TFIIB) and finally 4th TFIIH Result: Pre-Initiation Complex Note that RNA pol II does NOT bind the core promoter elements directly

Place the following events in order, as they take place during the process of gene expression (transcription and translation): 1) fMet-tRNA (initiator tRNA) binding to P-site, (2) Transcription of mRNA by RNA Polymerase, (3) Ribosome movement to the next codon, (4) Peptide bond formation, (5) Charged-tRNA, carrying amino acid, binding to a site on ribosome, (6) 30S ribosome subunit binding to mRNA

2,6,1,5,4,3

Approximately how many protein-coding genes are there in the human genome?

20,000

Consider the following segment of DNA: 5' CGGTCTTACCAGGTAGCA 3' 3' GCCACAATGGTCCATCGT 5' Assume the TOP strand is the template strand used by RNA polymerase. a)Draw the RNA transcribed b)Label its 5' and 3' ends . c)Draw the corresponding amino acid chain (refer to the codon table on the last page), assuming that the mRNA is translated in the first frame. d)Label its amino terminus.

3' GCCACAAUGGUCCAUCGU 5' 5'-UGC UAC CUG GUA ACA CCG -3' NH3-Cys Tyr Leu Val Thr Pro -COOH

Given the following anticodon sequence, draw a basic but accurate tRNA structure and include this anticodon in the structure. (2) What is the advantage to relaxing the base-pairing rules for the third position of the codon? In other words, why is wobble base pairing an advantage for a cell?

5' IAG 3' for full credit must show that the last 3 nucleotides at the 3' end are CCA Overall, fewer tRNAs are needed to pair with all the possible amino acid codong codons (there are 61)

How many protein-coding genes are in a typical yeast genome?

6,000

A) How does Gal4 bind to a specific DNA sequence (UAS)? Choose only that which leads to specificity. A. Hydrogen bond formation in the major groove B. Hydrogen bond formation in the minor groove C. Ionic bonds involving the negatively charged DNA backbone D. Covalent bonds involving the ribose of DNA B) The Gal4 protein is an example of which of the following: A. a general transcription factor B. a transcription activator protein C. a co-activator D. a RNA polymerase C) Which of the following proteins are recruited to the DNA when Gal4 is active? A. HAT B. HDAC C. Mediator D. RNA Polymerase II

A B A, C , D

Xist transcribed from the inactive X "coats" that chromosome leading to repression

A X inactivation center(Xic) is located on each X chromosome. • This Xic encodes along, non-coding RNA called Xist. • Counterintuitively, Xist is only transcribed from the inactive X (Xi). Xist is found to "coat" Xi.

CRISPR/Cas9 for genome editing 6 steps

A chemically synthesized RNA called the sgRNA targets a specific DNA sequence • The CRISPR/Cas9 complex, containing the sgRNA, identifies its target. • The sgRNA and target form a ~ 20 nt RNA/DNA hybrid, opens the dsDNA • Cas9 cleaves both DNA strands within the unpaired DNA sequence • Generates a ds DNA break • ds DNA break triggers DNA repair mechanism in cell

Translesion DNA synthesis

A damage-tolerance mechanism in eukaryotes that uses bypass polymerases to replicate DNA past a site of damage

Site on ribosome to which incoming charged tRNA is localized

A site

In nature, the CRISPR/Cas system is a kind of bacterial immune system against foreign genetic elements (DNA sequences) such as those from plasmids and phages (viruses). A) What is the purpose of CRISPR/Cas9 when researchers use it in the lab? B) Briefly explain how the system works to produce the desired outcome (described in your answer to part A) C) Relate your answer in part B to the original statement that in nature, CRISPR/Cas confers resistance against foreign genetic elements. In other words, how does that work?

A) What is the purpose of CRISPR/Cas9 when researchers use it in the lab? (2) To introduce mutations at a specific location on DNA B) Briefly explain how the system works to produce the desired outcome (described in your answer to part A). (3) A guide RNA binds via complimentary base pairing to a specific DNA sequence, marking that sequence with a RNA/DNA hybrid Cas9 goes to the marked sequence and cuts both strands of DNA Some change happens at the cut sequence with the result being the desired DNA alteration C) Relate your answer in part B to the original statement that in nature, CRISPR/Cas confers resistance against foreign genetic elements. In other words, how does that work? (2) The system can bind to specific sequences on the foreign DNA and mark it for cutting/destruction

Listed below are 2 important modifications associated with eukaryotic genomes. For each say whether it is associated with activation or repression. Use one or two sentences to defend your choice. Your defense should include mention of any writers or readers associated with each modification. A) lysine acetylation- Activation(1) B) Histone3 lysine 9 methylation-Repression(1).

A) lysine acetylation- Activation(1). Lysines on histone tails are acetylated by Histone Acetyl Transferases (HATs)that are recruited by activator proteins. (1) Acetylation loosens chromatin structure by neutralizing positive charge that is used in histone/DNA and histone/histone interactions. It also provides docking sites for components of the transcriptional apparatus (1). B) Histone3 lysine 9 methylation-Repression(1). H3K9 methylation is put on by a histone methyltransferase (SuVAR39H1 and is bound by HP1, which in turn binds suVAR39h1(1).This allow propagation of constitutive heterochromatin which prevents gene transcription.(1)

A figure of a simplified pre-initiation complex is shown below A) write the order of assembly of the factors shown. B) list one function for TFIID, one function of RNApol II and two functions of TFIIH

A) write the order of assembly of the factors shown. TFIID, TFIIB, RNA Pol II, TFIIH B) list one function for TFIID, one function of RNApol II and two functions of TFIIH. -TFIID binds the TATA sequence (specifically TBP) -RNA Pol II catalyzes the formation of phosphodiester bonds between RNA monomers -TFIIH (1) phosphorylates RNA Pol II CTD and (2) acts as a helicase to open up the transcription bubble

Below are the structures of lysine in its unmodified, acetylated, and tri-methylated forms(you will NOT need to memorize these structures for an exam). What are the functional consequences of acetylating and methylating lysine in histone tails for the transcription of eukaryotic genes? Explain how the structures shown below help explain their corresponding functions.

Acetylation of lysine neutralizes its positive charge and provides a binding site for proteins. Neutralization of the positive charge results in a less compact chromatin structure by disrupting histone/DNAinteractions. Methylation of lysine provides binding sites for histone code readers, which may activate or repress gene transcription.6.

CRISPR/Cas9 & Genome Editing CRISPR/Cas9 is a an easily engineered sequence- specific nuclease for use in eukaryotic cells! Efficient & precise tool for genome editing Can be used to: 3

Address fundamental research questions in many organisms • Introduce new or improved traits into plants • Gene therapy in mammals

Which of the following components involved in protein translation is responsible for "knowing" the genetic code (in other words: connects the amino acid code to the nucleotide code)?

Amino-acyl tRNA synthetases

Open Reading Frame (ORF)

An Open Reading Frame (ORF) is a long string of amino acid-specifying codons that begins with an AUG codon and is not interrupted by stop codons. Sequence of a DNA fragment with a protein coding sequence

The Ribosome is a Ribozyme Which parts of the ribosome are responsible for peptidyl transferase activity?

An RNA-only active site - there are no proteins within 20 Angstroms of the site of peptide bond formation, implicating rRNA as a fibozyme

Transfer RNA = tRNA

An adaptor molecule: brings AAs corresponding to each codon to ribosome • Small; 73-93 nucleotides (nt) • Conserved cloverleaf 2-D structure • Presence of modified bases, including inosine (I) and pseudouridine (y) • *Amino acid attachment at the 3 end of the tRNA at the end of the CCA-3 stem* • *Anticodon loop interacts with complementary codons on the mRNA by base-pairing* • Note directionality of anticodon

What does an E. coli promoter look like?

An alignment of DNA sequences in E.coli promoters reveal consensus sequences upstream of TSS (+1) Promoter strength correlates with the degree to which the promoter sequence matches the consensus

How does an epigenetically silenced gene differ from a mutant gene (a null, i.e. non-functioning, version of the same gene)?

An epigenetically silenced gene is non-functional in the sense that none of its gene product is produced. The lack of production is a consequence of the gene being "packaged" into chromatin that is unable to be transcribed, either because its transcriptional activators can't access the DNA or because they are unable to make the gene promoter accessible to the general transcription machinery. The gene sequence is the same as that of a gene copy that is normally expressed. A mutant gene has a change in the DNA sequence of the gene, either in its regulatory elementsor in its coding region, that prevents the normal expression of the protein or the activity of the protein if it is expression is normal.

Non-Homologous End Joining-NHEJ

An error-prone mechanism of double-stranded DNA break repair in eukaryotic genomes in which damaged nucleotides are removed and blunt ends of strands are joined. - result in mutant sequence

Mutation:

Any alteration in the genetic material that can be inherited

Describe the basic composition of the human genome using the following terms in your answer: exons, repetitive vs unique sequences, and total approximate size in bp.

Approximately 3 billion bp, 1.5% exons, and 50% repetitive and 50% unique sequences

Explain the statement: "Aminoacyl-tRNA synthetases are the only components of gene expression that interpret the genetic code."

As discussed in class, tRNA synthetases are the enzymes in the translation process that are responsible for connecting the nucleotide code to the amino acid code. Translation is really accomplished by matching the anti-codon sequence with the correct amino acid added to the 3' end of the tRNA, and tRNA synthetases are solely responsible for that task.

Which of the following would be disrupted if the CTD of RNA Polymerase II was unable to be phosphorylated. (Choose ALL that apply and do not guess because points will be subtracted for incorrect answers.) (2) A. The DNA strands would not separate to form a transcription bubble. B. RNA polymerase would not take on an "open" conformation capable of transcription. C. Proper chromatin modifications upstream and downstream of the transcription machinery would not occur during elongation. D. The resulting mRNA would be unstable because no 5' cap could be added.

B, C, D

Watson-Crick base-pairing interactions between RNA molecules plays an important role in many biological processes and experimental approaches to studying those processes. For each of the following processes or experimental approaches, give an example of such a pairing interaction along with a brief description of how that interaction contributes to the successful execution of the process. Bacterial translation initiation Eukaryotic translation regulation mRNA intron splicing Reverse genetic analysis of eukaryotic gene function

Bacterial translation initiation-Small subunit 16S RNA pairs with SD sequence to recruit small subunit to AUG Eukaryotic translation regulation-miRNA imperfectly pairs with sequence in 3'UTR to repress translation mRNA intron splicing-Multiple possibilities. U1RNA/5' splice site, U2 RNA to branch point consensus, U6 RNA to 5' splice site, U2 RNA to U6 RNA, U5 RNA to exon sequences. Each of these interaction organizes the spliceosome to promote catalysis of intron splicing Reverse genetic analysis of eukaryotic gene function-perfect pairing of siRNA to selected site in mRNA to cause cleavage and subsequent loss of mRNA function.

Bacterial vs. Eukaryotic Regulation

Bacterial: Ground state is on. Template for transcription is protein-free. Activators enhance weak polymerase binding; repressors interfere with polymerase binding. Polymerase holoenzyme binds protein-free DNA. *Promoters are DNA*. Eukaryotes: Ground state is off. Template for transcription is chromatin which is inaccessible to RNA polymerase. Activators make chromatin accessible and/or promote the formation of a marked site on the DNA. Repressors interfere with transcription by blocking activators or by making chromatin less accessible. RNA polymerase binds to protein-DNA complexes which mark the DNA. *Promoters are really protein-DNA complexes embedded in a chromatin environment*.

Regulation of transcription initiation: 3

Basal Expression: In the absence of both an *activator* and a *repressor*, RNA polymerase only binds occasionally to the promoter. Expression at low level. Negative Regulation: Binding of a *repressor* blocks binding of RNA pol, inhibits transcription. Positive Regulation: Binding of an *activator* recruits RNA Pol. Expression is activated, high levels of expression. E. coli lac operon regulated by an activator and a repressor working in this way

Ribosomes read the instructions for protein synthesis on mRNA - how it function - polarity

Bind mRNA and tRNA-AA • Positions them properly • Three tRNA binding sites: A: Aminoacyl/Acceptor P: Peptidyl E: Exit POLARITY: • The mRNA sequence is read 5 --> 3 • The amino acid chain is polymerized from N-terminus (made first) to C-terminus

TATA box

Binds a subunit of TFIID called TBP (TATA binding protein) Adjacent to transcription start site, required to get RNA polymerase onto promoter, sufficient in vitro on chromatin free DNA, may be present in different combinations on individual promoters. (Think of them as equivalent to -10 and -35 for bacteria.) A promoter DNA sequence crucial in forming the transcription initiation complex.

Splicing can be regulated by proteins binding to mRNA and spliceosomes

Branch point and 3' splice site bind to U2 snRNP and Protein Complex (PC), respectively Additional Regulation: • Repressor - RNA binding proteins that bind exons or introns at or near consensus sequence elements and interfere with spliceosome assembly. • Activator - RNA binding proteins that bind to "enhancer" elements in exons or introns and promote spliceosome assembly at intron/exon boundaries with "poor" 3' or 5' splicing sequence elements.

D) Focusing now on the lower diagram, the Mig1 protein is an example of which of the following: A. a chromatin "reader" B. another name for Mediator C. a transcription repressor protein D. a co-repressor E) Which of the following types of proteins would you expect Mig1 to recruit to the DNA? A. HAT B. HDAC C. Mediator D. RNA Polymerase II F) Gal4 effectively makes the chromatin _____ accessible to transcription machinery in the presence of galactose, and Mig1 effectively makes the chromatin _____ accessible to transcription machinery in the presence of glucose. A. more; more B. more; less C. less; more D. less; less

C B B

System used to edit eukaryotic genomes

CRISPR/cas9

Examples of alternative splicing in humans

Calcitonin/CGRP gene a) Calcitonin - binds to GPCR*; decreases blood calcium. b) Calcitonin Gene-Related Protein (CGRP) - binds to GPCR*; elevates cAMP in target cell

Redundancy of the genetic code:

Can be read out by a combination of wobble base-pairing AND • Presence of multiple independent tRNA molecules with different anticodons carrying the same amino acid • Example: Alanine • 4 codons, but not 4 tRNAs

An aminoacyl-tRNA linkage

Carboxyl group of amino acid is linked to 3' OH of tRNA

CRISPR- associated protein 9 nuclease

Cas9

developmentally regulated X chromosome inactivation - Coat pigmentation in tortoiseshell cats

Choice of X chromosome for inactivation occurs early in development, and cells that arise from each early cell have the same X inactivated. Thus the patchy fur color. Xist transcribed from the inactive X "coats" that chromosome leading to repression

How does ribosome know where to start?

Choice of the AUG initiator codon in prokaryotes depends on base- pairing between the 16S rRNA and *the shine dalgarno* (also called the ribosome binding site or RBS) on the mRNA (3-9 bp interactions)

Mediator

Coactivator

Ribosomes - The big picture:

Coupled transcription & translation in prokaryotes Polysomes = multiple ribosomes translating a single mRNA transcript

What is meant by the statement: "Bacterial translation and transcription are tightly coupled"?

Coupling means that translation of an ORF in a bacterial mRNA can be translated as soon as the RNA polymerase has synthesized it and made available its Shine-Dalgarno sequence and initiator AUG codon. These two processes are not separated in space nor time in bacterial cells.

Sequence-specific transcription activator proteins have distinct ___

DNA binding and activation domains which are interchangeable

wobble

Deviations from standard Watson-Crick base- pairing rules in codon-anticodon interactions at the *3position of the codon* Flexibility in the base-pairing rules in which the nucleotide at the 5' end of a tRNA anticodon can form hydrogen bonds with more than one kind of base in the third position of a codon.

An E. coli genome fragment encodes many genes (many operons)

E. coli genome: Density of genes is very high • 4.6 million bp (megabasepairs) • 4377 genes, *many in operons, both strands used* • Essentially no introns

fMet-tRNAf, the initiator tRNA and Met-tRNA are not the same

EF-Tu does *not* associate with fMet-tRNAf, the initiator tRNA • Only IF2 can functionally interact with fMet-tRNAf • EF-Tu delivers Met-tRNAm to AUG codons within the ORF during the elongation process

tRNA/tRNA Synthetase Recognition

Each amino-acyl-tRNA synthetase has binding pockets for a specific AA and its cognate tRNA

Termination of Translation in Prokaryotes

Elongation cycle continues until a stop codon is encountered • Termination mediated by protein Release Factors (RF) that interact with stop codons in the A site • RF structurally resemble tRNA (see next slide) • Probably allow water access to ester bond between AA and tRNA in P site • Ribosome dissociates, tRNAs and completed polypeptide are released

The Genetic Code is Universal (with very rare deviations) Enables genetic engineering

Enables genetic engineering A protein coding sequence (string of codons) from one organism can be transferred to another organism to encode the production of the same protein

Translation in eukaryotes starts by the ribosome recognizing the 5' Cap. What key difference between transcription/translation in prokaryotes and eukaryotes accounts for this difference in how translation starts?

Eukaryotes do not have polycistronic mRNA, like prokaryotes do. So in Eukaryotes there is only one start codon per mRNA, and it is close to the 5' end of the message. Prokaryotes need to be able to identify start codons not near the 5' end of the mRNA.

Histone Modifications at the Promoter Regulate

Eukaryotic Transcription In Vivo

Many prokaryotic genes are expressed as multi-gene operons from "polycistronic" mRNAs. Eukaryotic genes are almost always expressed as single genes from "monocistronic" mRNAs. What property of the eukaryotic translation machinery accounts for this difference?

Eukaryotic translation is started by recruitment of small ribosomal subunits to 5'Cap bound initiation factors(2). The small subunits then scan down the mRNA to the first AUG where they stop, recruit the large subunit and begin (2). There is no way for Cap bound complexes to get to AUG initiation codons for downstream ORFs in a polycistronic mRNA

Recall F' plasmid & partial diploids

F' plasmid with 2nd copy of small DNA segment • For example: Add 2nd copy of lacI gene • This allows scientists to examine dominant/recessive relationships between alleles, and to ask if relative position of alleles is important

The s subunit of the holoenzyme contacts the -10 and -35 regions • Fits in

Fits in major groove

Based on our description of galactose gene transcription in yeast, inactivating mutations in which of these "Gal" genes would result in yeast mutants which would be unable to grow on galactose. Explain your reasoning. GAL1, GAL4, GAL80, GAL3.

GAL1-unable to grow. Gal1 codes for an enzyme involved in metabolizing galactose; lack of the enzyme would prevent yeast from growing on galactose. GAL4-unable to grow. Gal4 codes for a transcriptional activator protein that binds to sites upstream of genes required for growth on galactose and activates their transcription in the presence of galactose. Without gene activation, the enzyme won't be made that is needed to metabolize galactose. GAL80-able to grow. Gal80 binds to and inhibits the activation domain of Gal4 in the absence of galactose. Its absence would result in constitutive (unregulated) synthesis of enzymes used to metabolize galactose; wasteful but not growth inhibitory in the presence of galactose. GAL3-unable to grow. Gal3 binds to galactose to inhibit Gal80 inhibition of Gal4. In the absence of Gal3, galactose cannot"induce" Gal4 activity, so the gene will not be transcribed, the enzyme will not be made, and galactose cannot be metabolized.

Activator of Yeast Gal1 gene transcription

Gal4

In the absence of chromatin, what transcriptional machinery do eukaryotes require?

General Transcription Factors AKA: GTF's

A Mammalian RNA Pol II gene has diverse regulatory regions

General transcription factors (GTF's) bind to the core promoter Regulatory transcription factors (a.k.a. regulator, activator, repressor) bind to enhancers or nearby elements

Sickle Cell Disease & Gene Therapy

Genetic diseases involving haemoglobin, such as sickle cell disease (SCD), are caused by mutations in the β-globin (HBB) gene. • SCD is caused by a single nucleotide mutation (A to T), which changes the normal Glu at amino acid at codon 6 the HBB gene29 to Val (Glu6Val). • Mutation results in a change in the shape of red blood cells, which results in clumping • Gene editing in patient-derived haematopoietic stem cells followed by autologous transplantation could potentially be used to cure β-haemoglobinopathies

Structure that triggers rho-independent termination of transcription

Hair pin

Problem: A core promoter in chromatin is inactive. How do you make it accessible to GTFs and RNA polymerase?

Histone Acetylation "Loosens" Chromatin Nucleosome remodeling Chromatin remodelers

List the targets of activation domains of DNA-binding transcription activator proteins.

Histone code "writers" (HATs and certain HMTs), Mediator, TFIID and Chromatin Remodeling Complexes

Local Repression by a protein regulator (temporarily off)

Histone deactylases associate with transcriptional co-repressors - JSYK: --- Just as DNA-binding activators can bind to co-activators to promote transcription, DNA-binding repressors can bind to co- repressors to inhibit transcription. --- Recall that some activators recruit co-activators with histone acetylase (HAT) activity --- Similarly, some repressors recruit co- repressors have histone deacetylase (HDAC) activity. The repressor Mig1 recruits HDAC to inhibit GAL1 transcription in the presence of glucose. --- In yeast, glucose availability induces the binding of a repressor called Mig1, which recruits a co-repressor with HDAC activity. --- In human cells E2F is a transcription factor (regulator protein) that controls genes involved in cell-cycle progression and binds to the RB protein. E2F can function as an activator or a repressor, depending on whether it associates with RB, co-repressor complex with HDAC activity, or a co- activator with HAT activity.

DNA/Histone Interactions:

Histones interact with the DNA minor groove; A/T base pairs are favored. Implication: *sequence- specific positioning of nucleosomes on DNA*. Histone amino-terminal "tails" are conserved (but are not part of the "core") and are targets for covalent modifications that regulate chromatin structure and provide binding sites for proteins

RNA Processing (in Eukaryotic Cells)

I. 5' mRNA capping II. Cleavage and polyadenylation ("poly A") addition to generate the 3' end of the mRNA III. Intron splicing (removal of intron sequences)

Genetic approaches for investigating gene function: 2

I. Introduction of extra or altered versions of genes into the genome (transgenic approach) II. Reverse genetics & Genome editing

In prokaryotic translation, brings fMet-tRNA to the start site

IF2

Epigeneticists believe that DNA is marked during early development, but that marks can be erased or reprogrammed later in life. Propose some kind of treatment/substance that might result in removal of epigenetic marks, and explain the reasoning behind your answer.

If a person/animal was treated to reduce the availability of methyl groups or something that specifically removes methyl groups from methylated DNA, that might erase epigenetics marks

Uses/ Applications for CRISPR/Cas9

Imprecise insertions or deletions from Non-homologous End Joining (NHEJ) --> often loss of function • Precise changes in nucleotide sequence through homology directed repair (HDR) • Desired new sequence (donor DNA) must be provided • Example: correction of the sickle cell mutation in haemoglobin gene

intron and exons

In general, introns are large and exons are small. in humans, intron represent the majority of sequence of the gene

E2F transcription factor

In human cells E2F is a transcription factor (regulator protein) that controls genes involved in cell-cycle progression and binds to the RB protein. E2F can function as an activator or a repressor, depending on whether it associates with RB, co-repressor complex with HDAC activity, OR a co- activator with HAT activity. --- KSYK example: Here RB is not phosphorylated, which results in association with an HDAC. The result is repression of transcription of cell cycle regulated genes. protein that, when not bound to the Rb protein, activates the transcription of genes coding for proteins required for DNA replication and entrance into S phase of the cell cycle

How do regulatory proteins sense presence of inducer?

Inducers (e.g. lactose) bind to repressor protein at a second site, change ability of protein to bind to DNA.

Base Excision Repair

Initiated by an enzyme that *recognizes* a damaged base and *removes* it. Example: Modified base is a deaminated C which is a U.

Make a list of all the protein factors involved in translation that we discussed in lecture (Initiation Factors, Elongation Factors and Release Factor). For each protein factor describe its role in the translation cycle. Make note if the protein is a G-protein, and indicate how this impacts the function of the factor.

Initiation Factors: •IF3 (you don't need to memorize this protein name) prevents the premature assembly of the ribosome. IF3 binds to the 30S su and prevents the 50S su from binding until the 30 S Initiation complex has been formed. •IF2 binds to the initiator tRNA charged with formyl methionine (fMet), and brings it to the P site on the 30S subunit. This forms the 30S initiation complex, and establishes the correct ORF for the protein. IF2 is a G-protein. It is in the GTP-bound form when it brings tRNA-fMet to the P site. Upon binding of the 50S ribosome to the complex, GTP is hydrolyzed, and the IF2-GDP is released. Elongation Factors •EF-Tu is also a G-protein. EF-Tu in the GTP bound form binds to charged tRNAs, stabilizes the labile linkage between the amino acid and the 3'OH on the tRNA. EF-TU brings the charged tRNA to the A site of the ribosome. If the codon/anticodon sequences are complementary, the GTP on the EF-Tu is hydrolyzed, and the EF-Tu-GDP bound form leaves, with the correct tRNA loaded into the A site. •EF-G is another G-protein. It is also called the "translocase". It binds to the A site, displacing the tRNA in the A site, thus shifts the tRNA that was in the A site into the P site(Note: this tRNA now has the growing peptide attached to it). Hydrolysis of the GTP on EF-G provides the energy required for EF-G to insert itself into the A site, forcing the mRNA and its bound tRNAs to move over one codon in the ribosome Release Factors: •Release factors (RF1 and RF2) are involved in terminating translation. •This class of RFbind to stop codon positions in the A site, and in doing so promote the hydrolysis of the acyl bond between the final amino acid on the nascent peptide the 3-OH of the tRNA in the P site.

Inosine and the "wobble" position

Inosine(I) is a deamination product of A, made after tRNA transcription • I is present in the anticodon loop of some tRNA molecules • I base-pairs with U, C or A in the wobble position (3 position)

Termination of Transcription: 1 ) Intrinsic mechanism

Intrinsic mechanism: •Termination sequence: G-C rich region followed by string of A's. •After transcription though 3' UTR RNA forms hairpin loop •Triggers release of completed transcript and RNA polymerase from DNA

Sources of DNA Damage

Ionizing radiation - X-rays; cosmic rays; radon Result: single and double-strand DNA breaks Ultraviolet (UV) radiation - sun exposure Result: pyrimidine dimers (photodimers), C to U conversion Chemicals - chemotherapy reagents, psoralens, aflotoxins Result: damaged bases, cross-links Oxidation - during the course of normal cell metabolism

negative control: Repression of the lac operon in the absence of lactose (inducer) Lac Repressor proteins bind to ____ and prevent RNA polymerase from binding to ________.

Lac Repressor proteins bind to *lacO* and prevent RNA polymerase from binding to *promoter*. Inhibits transcription of lac operon

In class we discussed three protein factors that participate in bacterial translation that bind and hydrolyze GTP during the course of their function. A) List these three factors B) Imagine a mutation in which the GTP-ase activity of EF-G is impaired, so that it is no longer able to hydrolyze GTP. What would the effect of this mutation be on translation? Explain your answer

List these three factors(1pteach) IF-2, EF-Tu, EF-G This mutation would stop protein translation, or at least slow synthesis/elongation (1) Explanation: without being to hydrolyze GTP to GDP, EF-G would not be able to insert itself into the A site of the ribosome (1). The hydrolysis of the GTP provides the energy required for this process. Thus, the tRNA in the A site would not be translocated into the P site, and the ribosome would be stuck (1 pt)

For each of the following terms, indicate whether it is associated with gene activation, gene repression, or both. Give a one sentence defense of each answer. Mediator DNA methylation Chromatin remodeler Histone acetylation Heterochromatin

Mediator -Activation -it is a complex that binds to both activator proteins and RNA polymerase. DNA methylation -Repression, methylation at C5 position of cytosine, provides a docking site for Histone deacetylase which promotes repressed chromatin state. Chromatinremodeler -Activation-when remodeling makes a promoter more accessible to RNA polymerase. Histone acetylation -Activation-neutralizes lysine charge leading to less condensed chromatin. Also provides binding sites for Readers that are part of the transcriptional activation machinery. Heterochromatin -Repression-condensed chromatin state resulting from specific histone methylation marks (H3K9 mostly), which are read by factors that set up the repressed state.

What additional elements are required for transcription when the DNA is part of chromatin in vivo?

Mediator is not required for basal transcription in vitro but it is also considered to be a GTF

DNA methylation of eukaryotic DNA is associated with gene repression. Where are methyl groups added to DNA and what is one mechanism by which this modification results in gene repression?

Methylation of DNA occurs at the 5 position of the cytidine ring in cytosines located within CpG dinucleotide sequences in eukaryotic DNA. One way in which this modification promotes gene repression is by binding Methyl Binding Domain (MBD)-containing proteins that recruit histone deacetylases to the chromatin harboring the methylated DNA.

When referring to a eukaryotic gene, what is meant by the term "core promoter" ?

Minimal set of sequences needed to recruit RNA polymerase and the general transcription factors to DNA and to get transcription initiation at a specific site on the DNA in a chromatin-free test-tube environment.

What is a gene?

Molecular definition: All information needed to synthesize RNA or protein (not all genes encode proteins) • Promoter: determines where transcription starts • Regulatory sequences: when, where, how much • Coding sequence: Open Reading Frame, ORF, if gene encodes protein • Transcription terminator: where transcription should end Genetic Definition • Everything required to add back to a mutant to restore function of the gene (complementation).

Alternative splicing patterns: One gene can code for multiple proteins depending on the specific intron splicing pattern

More than 50% of human genes are subject to alternative splicing and/or RNA processing. What determines how exons are spliced together?

Mismatch DNA Repair steps

MutS recognizes the mismatch (or small deletion or insertion). MutH recognizes the methylated parent DNA strand, binds to the half methylated site nearby and nicks the unmethylated, newly synthesized DNA strand. MutH is an endonuclease. The mutant strand is removed by exonuclease 1 digestion, starting from the nick generated by MutH. DNA polymerase replaces the missing DNA with the correct nucleotide incorporated. The nick is sealed by DNA ligase.

Type of DNA repair mechanism that usually results in loss of gene function

NHEJ

Explain 2 reasonswhy the 16S rRNA gene is a good choice when using a metagenomic approach to characterize a microbiome

Need two of the following: •The 16S rRNA gene is present and well conserved in all bacteria, and so is a good choice as a general diagnostic gene for monitoring which taxa are present in a sample. •Regions of the 16S rRNA gene are highly conserved (e.g. the 3' and 5' ends), so that universal primers can be used to amplify the gene from essentially all bacteria (see slide from 4/24 lecture). •The 16S rRNA gene also contains variable regions, that are evolving at a relatively quick rate that can be used to distinguish different taxa from each other. •Additionally, since the 16S DNA sequence is known for many well-characterized (e.g. reference) organisms, comparing new sequences to known sequences can tell us a lot about the taxonomy of an organism, how closely related it may be to known, characterized bacteria.

Part of a gene that directly codes for protein

ORF

Classic Example of a Bacterial Operon: The Lac Operon of E. coli Operon: Two or more adjacent __________that are coordinately controlled by transcription from a single ________ • A single mRNA that harbors multiple protein coding sequences is called a ____________ mRNA

Operon: Two or more adjacent *structural genes* that are coordinately controlled by transcription from a single *promoter* • A single mRNA that harbors multiple protein coding sequences is called a *polycistronic mRNA*

The lac operator = lac repressor binding site - characteristic:

Palindromic - two-fold (dyad) symmetry

Jacob & Monod used genetics to discover and study the lac operon, part 2 Phenotypes of lac operon mutants

Phenotypes of lac operon mutants • lacZ -, lacY - mutants: disrupted proteins, no activity • lacP mutants: promoter impaired, less or no activity • lacI - mutant: lacZYA genes not regulated by lactose, constitutively expressed • lacOc mutant: lacZYA not regulated by lactose, constitutively expressed

Together, general transcription factors (GTF's) and RNA polymerase assemble at the core promoter to form a

Pre-Initiation Complex (PIC)

A Mammalian Pol II Gene

Promoter-Sequences adjacent to transcription start point; determines where transcription starts. The set of sequences required to recruit General Transcription Factors & RNA Polymerase *to non-chromatin DNA in a test tube* is called the *Core Promoter*. Promoter-Core promoter plus nearby elements. ^^Minimal sequence required to recruit RNA polymerase to transcription start site *in a cell (i.e. to DNA packaged into chromatin*) Enhancer Elements: *Enhancers* work from far away to increase transcription; They are needed for maximal promoter activity, and often involved in cell-type specific expression (for example, liver, muscle, or gut

Mammalian PolII Gene

Promoter-Sequences adjacent to transcription start point; determines where transcription starts. The set of sequences required to recruit General Transcription Factors & RNA Polymerase to non-chromatin DNA in a test tube is called the Core Promoter.

Modified histones are differentially associated with promoters and transcription units

Promoters are enriched in acetylated histones. • Transcribed chromatin is enriched in H3K36 methylated histones. A HAT, and a histone methyl transferase (HMT), bind to the phosphorylated RNAP CTD tail. The HAT, a writer, activates chromatin as the polymerase transcribes. The HMT writer then methylates histones at H3K36, which recruit a histone deacetylase (HDAC). The HDAC erases the acetyl marks to inactivate the chromatin after polymerase has passed through.

How can a RNA (or DNA) sequence determine an amino acid sequence?

RNA linear code = 4 letters (A, C, G, U) --> Translation --> Protein linear code = 20 letters (amino acids)

More complex transcriptional regulation in eukaryotes: •Three RNA polymerases

RNA pol I - rRNA *RNA pol II - mRNA & snRNA* RNA pol III - tRNA & 5s rRNA

CAP activates by recruiting

RNA polymerase DNA-bound cAMP-CAP recruits RNAP by binding C-terminal domain (CTD) of the a subunit

Elongation of RNA transcripts

RNA polymerization does not require a primer • 5' end of prokaryotic transcript has rNTP • Reaction driven by hydrolysis of PPi

Type 1 Self-splicing intron Formation of peptide bond is catalyzed by a

RNA with catalytic activity (Ribozyme)

Activation of most eukaryotic genes requires the activity of complexes that carry out the process of nucleosome or chromatin remodeling. What is meant by "remodeling" and how is remodeling activity localized to specific sites on the genome?

Remodelers use the energy of ATP hydrolysis (1)to reposition DNA within nucleosomes cylindrical surface, making sequences more (or less) accessible to the transcription machinery(1). Remodelers are located to specific sites by interacting with the activation domains of factors (1)that have DNA binding domains that bind to specific sequences

What is reverse genetics and why would a scientist use it?

Reverse genetics is when a gene sequence is known, and than a mutation in this gene is introduced into the organism to generate a mutation. Characterization of the mutant phenotype provides information about the function of the gene. Reverse genetics can also be used to change the function of a gene, edit a gene for gene therapy, or to change the expression of a gene.

Initiation of Transcription -- Roles of subunits

Roles of subunits a: enzyme assembly, promote interaction with regulatory proteins b: catalysis b': binds DNA, catalysis s: positions holoenzyme to initiate trx: binds to -10 & -35 regions of promoter. s : dissociates from RNA Pol after elongation begins Focus on sigma

Proteins that bind exons and recruit splicing machinery

SRs

Why is it important to be able to characterize microbial communities using culture-independent techniques like metagenomics?

Scientists have not been able to culture most microbes in the lab, so if they were to rely simply on culture-dependent methods, they would miss most of the microbes present in various environments and communities.

In the Whole Genome Shotgun Sequencing method, a genome is fragmented, sequenced, then reassembled. Describe both why fragmentation is necessary and how the genome sequence is reassembled.

Sequencing techniques are limited in the length of DNA strand that can be sequenced (DNA sequence quality decreases with sequence length). The sequences are reassembled by matching (aligning) overlapping fragments. This step is largely computerized.

Compare the two DNA repair mechanisms (NHEJ and homology-dependent repair) that can occur in CRISPR-Cas9 gene editing. Briefly explain each mechanism, how they differ, and what influences a researcher to select each method.

Similarities = both methods repair double-stranded DNA breaks Differences = NHEJ usually leads to insertions and deletions and loss of function of the gene at that location while homology-dependent repair can use a donor piece of DNA as a template, and thus DNA maintaining the original sequence without introducing a deletion or insertion. Researchers may use NHEJ when the goal is to knock out a gene of interest and study the results of the lack of gene function. Researchers use homology dependent repair when the goal is to precisely edit or repair a gene.

tRNA anticodon : codon interactions

Some tRNAs must recognize multiple codons (Ala has 4) • Inosine (I) is present in the anticodon loop of some tRNA molecules • I is a deamination product of A, made after tRNA transcription • *I base-pairs with A, U or C in the wobble position*

How spliceosomes work:

Splicing begins with recognition of the 5' splice site by the 165 base long U1 snRNA found in the U1 snRNP.

Starch Amylose: Amylopectin

Starch: a polymer of glucose • Branching pattern impacts the properties of starch. • Amylose: mostly unbranched chains, more resistant to digestion, not soluble in cold water. • Amylopectin: short, highly branched chains. Higher levels of amylopectin result in a more sticky "glutinous" or "waxy" starch. • Starch high in amylopectin has properties useful to paper, cardboard, textile and adhesive industries

Classic Example of a Bacterial Operon: The Lac Operon of E. coli Structural genes encode b-galactosidase, Permease, Transacetylase

Structural genes encode b-galactosidase, Permease, Transacetylase • b-galactosidase : lactoseàglucose + galactose • Regulatory DNA at 5' end: Promoter (P) and Operator(O) • Adjacent gene encodes LacI = Lac Repressor. • Lac Repressor always present in low levels • Binds to lacO site on DNA • Binds to lactose (the inducer) • When bound to lactose Repressor doesn't bind lacO

A promoter sequence in eukaryotic transcription

TATA box

CRISPR/Cas9 used to edit β-haemoglobin gene in Human haematopoietic stem cells

Tested whether the HBB-edited haematopoietic stem cells (HSCs) still able to differentiate into RBCs, could express haemoglobin (Hb). • Observed presence of mature differentiated erythrocytes that expressed Hb (based on RNA levels) • Targeting HSC with corrective SNP donor reverted an average of 50% of the Glu6Val (HbS) alleles to wild-type (HbA) alleles • Gene editing in patient-derived HSCs, followed by autologous transplantation could potentially be used to cure β- haemoglobinopathies • System still needs optimization: off-target mutations can occur

Addition of a poly(A) tail to the 3'end of pre-mRNA

The "context" (position) of the cleavage signal is important. The functions of the polyA tail are not firmly established. The polyA tail may be involved with (a) stability of mRNA and (b) enhancing translation efficiency. - Blocking polyA synthesis does not interfere with the synthesis of the primary transcript. - mRNA that do not have a polyA tail can be transported out of the nucleus, however, protein synthesis is less efficient

Why are the requirements for getting transcription of DNA started different in a cell than in a test tube?

The DNA is in a more condensed state, called chromatin, when it is in a cell.

Transcription Regulation Due to Nutrient Availability in Yeast: A Case Study - genes - +/- of inducer

The GAL7, GAL10, andGAL1 genes in yeast are coordinately regulated by a transcriptional activator, Gal4. • Gal4 proteins bind to yeast "enhancers" (Upstream activation sequence, UAS) to activate transcription when galactose is present. • In the *absence of galactose*, *Gal80* protein binds to the *Gal4* activation domain. • While *Gal4* still binds to DNA, it can *no longer activate transcription.* In the *presence of galactose*, *galactose* binds to *Gal3* protein, which binds to *Gal80* in the cytoplasm. This prevents Gal80 from getting to the nucleus .Gal4's activation domain is now exposed to promote transcription initiation of GAL7, GAL10, and GAL1.

Considering translation in prokaryotes, explain, including a diagram, how the start codon gets correctly placed in the P-site of the ribosome to start translation. Be specific on how the translation machinery knows which AUG is the start codon

The Shine Dalgarno sequence on the mRNA makes complementary base pairing with the 16srRNA that is part of the small subunit of the prokaryotic ribosome. There is set spacing between the Shine Dalgarno sequence and where the P site is available upon large subunit. The AUG that falls in that correct spacing is the start codon.

What role does the Shine-Dalgarno sequence play in bacterial translation? How does the use of the Shine-Dalgarno sequence allow the existence of polycistronic mRNAs in bacteria? As part of your answer, draw an mRNA for an operon, in which there are three open reading frames (ORFs). On your diagram indicate where the Shine-Dalgarno sequences are in the operon.

The Shine-Dalgarno (SD) sequence is a short consensus sequence that lies 5-13 bases upstream of the first AUG of the open reading frame(ORF)of bacterial genes. It recruits the small (30S) ribosomal subunit to the first AUG by base-pairing with a complementary sequence near the 3'end of the 16S rRNA. Ribosome binding to the SD sequence only requires that it be accessible, not that it be near the 5' end of the message. Downstream ORFs in polycistronic mRNAs need only have good SD sequences downstream of the stop codon from the ORF before it and be located at the appropriate distances upstream of the AUG initiation codons to be efficiently translated

How is the genetic code read?

The amino acids themselves are unable to read the genetic code • The genetic code is read by: • transfer RNA (tRNA)molecules • aminoacyl-tRNA synthetases • ribosomes

NUCLEOSOMES & CHROMATIN

The average human chromosome is 5 cm long, and The nucleus of a human cell is 5-10 microns. How does all the DNA fit in the nucleus? Lots of packing to reduce DNA volume All eukaryotic DNA is packaged into nucleosomes. DNA (~160base pairs) is wrapped around a protein core made of 8 histone molecules:

How do the enzymes recognize which strand is correct and which contains the mismatch?

The enzymes recognize the methylation on the template, "correct" strand. The newly synthesized strand hasn't had a chance to be methylated in the cell.

Compare and contrast the genetic information that is present in the DNA versus the information present in the primary transcript of a prokaryotic gene (e.g. the mRNA). What are the roles of the DNA that does not directly code for protein? Please include a drawing of a prokaryotic gene that helps explain your answer.

The gene includes all of the information required to express a functional RNA or mRNA (shown below). This includes: i) the regulatory regions (promoter, operator, any other bindings sites for transcriptional regulatory proteins) that regulate transcription, ii) 5' Untranslated region (UTR), iii) coding sequence (would be the open reading frame (ORF) for a protein coding region, iv) 3' UTR and v) transcription termination signal.The transcript contains less information. It starts at +1, the transcription start site, and includes the i) 5'UTR, ii) coding sequence (e.g ORF), and iii) 3'UTR. The sequences that provide regulatory information regarding transcription initiation, level of transcription, or termination, are not included in the RNA

Histone protein structures

The histone fold handshake: H2A/H2B & H3/H4

What structural and/or chemical features of DNA and DNA binding proteins account for the fact that most transcriptional activator and repressor proteins bind in the major groove of the DNA?

The relevant properties are the structures of alpha helices in DNA binding proteins, the most common protein structural motifs to interact with DNA, and the geometries and hydrogen bonding properties of base pairs in the major and minor grooves of B form DNA. The major groove is large enough to accommodate an alpha helix protein structure, the minor groove is not. The base pairs have unique hydrogen bonding patterns in the major groove, not in the minor groove, so this allows for specific amino acid-DNA nucleotide binding to take place.

What component of the CRISPR-Cas9 system can be designed by researchers to target a specific gene? Draw a figureCRISPR-cas9 complex binding to DNA and show where, approximately, the DNA is cut relative to the sgRNA annealing.

The sgRNA can be designed by researchers to anneal to a genome locus of interest The figure below shows the complex binding to DNA, and causing the ds DNA to open in the region to generate a small region of ss DNA. The scissors in the figure show the approximate location of the cuts to the DNA strands. Note: both ss DNA strands are cut.

Why is cancer often considered to be a genetic disease? Briefly describe two observations that illustrate the connection.

There are several observations that provide support for the claim that cancer is a genetic disease, including: a)Many mutagenic chemicals are also carcinogenic b)Many mutations in DNA repair-related genes lead to increased incidence of cancer. For example, patients with Xeroderma pigmentosum lack nucleotide excision repair.

Intron splicing (removal of intron sequences) - Splicing mechanism:

There are two steps in splicing, both are classified as trans-esterification reactions, where a phosphodiester bond is broken and remade. The reactions in intron splicing are mediated by a complex called the spliceosome

Jacob & Monod used genetics to discover and study the lac operon, Part 1 TOOLS:

Tools: • Wild-type E. coli: lactose induces expression of lac operon • 2 types of E. coli mutants: 1. In structural genes: lacY- (no permease), lacZ- (no b-gal) 2. Regulatory mutants: lacP, lac0, lacI • Partial diploids (2nd copy of small DNA segment on F' plasmid)

Mutations can result from failure to repair damaged DNA. Whyis mutation associated with cancer?

Tumors arise through a series of sequential mutational events that lead to a state of uncontrolled cell proliferation Uncontrolled cellular proliferation = unchecked cell division Invasiveness (metastasis)

With the advances in DNA sequencing methods, obtaining human sequence data has become much more accessible. Describe two examples of applications of human genome sequence data discussed in lecture.

Two of the following: Identification of human remains, identification of genes for determining health risk factors, human ancestry

Ribosome Composition in prokaryotes

Two subunits: • Large 50S • Small 30S • Each subunit contains both ribosomal RNA (rRNA) and many proteins [vvv: JSYK • 50S = 23S rRNA+ 5S rRNA + 31 proteins • 30S=16SrRNA +21 proteins] rRNA makes up most of ribosome structure Proteins are stabilizers. --They poke into crevices.

first component of the spliceosome recruited during intron excision

U1 snRNP

CTD (C-terminal domain)

Unphosphorylated carboxyl- terminal domain (of largest subunit) is a promoter tether. the domain of RNA polymerase that is involved in stimulating transcription by contact with regulatory proteins

The protein classes that "write", "erase", and "read" these histone modifications.

W- methyl or acetyl transferases E - de[methyl/acetyl]ases R- methyl or acetyl readers ---[M]: chromodomains --- [A]: bromodomains

Addition of an inducer stimulates production of an enzyme

When a new carbon source (e.g. lactose) is added to the growth medium of E. coli, synthesis of the proteins needed to take up & metabolize it is induced. • Usually regulated at the level of transcription: mRNA encoding the enzyme is induced • Regulatory mechanisms can be positive or negative

Structure of B-Form DNA

Wide, deep major groove - The major groove is not only accessible to DNA binding proteins but is also the most information-rich Good size to fit a-helix of a DNA binding protein Narrow, shallow minor groove

Deviations from standard rules of nucleotide base-pairing that allow some tRNAs to interact with multiple codons.

Wobble

Histone tail interacting protein classifications

Writers: Covalent modification of histone amino acids Erasers: Restore modified histone amino acids to unmodified form Readers: Bind modified histone amino acids An Example: Writer: Histone acetyltransferase (HAT) Activates transcription Eraser: Histone deacetylase (HDAC) Represses transcription

All DNA sequence elements including a coding region, transcription start and stop sites, promoter, and regulatory sequences

a gene

A charged tRNA

a transfer RNA molecule to which the appropriate amino acid has been attached

If you alter the sequence of the lacoperator site such that repressor can no longer bind, what do you expect will bethe consequences for lacoperon expression under the following growth conditions (growth medium with the following sugars added)? Explain your reasoning a)Medium no lactose, no glucose b)Medium plus lactose, no glucose c)Medium plus lactose, plus glucose

a)Medium no lactose, no glucose •High level expression: •No glucose, so cAMP levels are high, CAP/cAMPis active and can bind to the activator site torecruitof RNA pol •No repression, despite the absence of the inducer lactose, because no functional operator, so Repressor cannot bind. b)Medium plus lactose, no glucose •High level expression: •No glucose, so cAMP levels are high, CAP/cAMP recruitment of RNA pol •No repression because Repressor cannot bind (no functional operator). The presence or absence of lactose does not make a difference. c)Medium plus lactose, plus glucose •Low (basal) level expression: •Glucose present, so cAMP levels low, no CAP recruitment of RNA pol; therefore, only basal transcription •No repression because Repressor cannot bind (no functional operator).

Positive and Negative Regulation

a. In the absence of both an *activator and a repressor*, RNA polymerase only binds occasionally to the promoter. Expression at low (basal) level. b. Binding of a *repressor* prevent RNA pol from transcribing c. Binding of an *activator* recruits RNA Pol. Expression is activated, see high levels of expression. • E. coli lac operon regulated by an activator and a repressor working in this way

Gene mutation:

alteration in the nucleotide sequence of a gene

Chromosome mutation:

alterations of chromosome structure or chromosome number

The C-Terminal Domain (CTD) of Pol II is an ___ or ___ of the gene expression machinery during elongation

an integrator or coordinator

Protein-protein interaction allows two transcription factors to bind ___ to DNA

bind "cooperatively" to DNA. Cooperative means each binds better to its site on the DNA in the presence of the other.

which strand of genome can serve as templates for RNA synthesis

both strands

Transcriptional regulator proteins that bind to far-away enhancers are brought into the proximity of the promoter by

by DNA looping and interactions with Mediator.

Define the term "core promoter." List the components needed in a test tube in order to transcribe non-chromatin eukaryotic DNA.

"Core promoter" refers to the minimal set of DNA sequences required to recruit RNAPol II and initiate transcription of a gene when in a nonchromatin structure. The components needed are the "core promoter,"general transcription factors, and RNA Pol II.

List and define the function and binding order of the four general transcription factors discussed in lecture.

"DeBoRaH" TFIIDbinds to the promoter, TFIIBbinds to TFIID, then RNA PolII binds. Finally, TFIIHbinds and "opens" the DNA for transcription. This forms the "pre-initiation complex." TFIIH is both a helicase and a kinase. The kinase phosphorylates amino acids in the C-terminal domain (CTD) of RNA PolII subunit 1. Phosphorylation of the CTD untethers PolII. Helicase activity unwinds the duplex DNA.

histone modifications - "Erasers"

"Erasers" restore modified histone amino acids to unmodified form - de[methyl/acetyl]ases

histone modifications - "Readers"

"Readers" bind modified histone amino acids - methyl or acetyl readers ---[M]: chromodomains --- [A]: bromodomains

A histone methylation "reader", HP1, helps heterochromatin spread by recruiting HMT 4 steps

1. Histone methylase(HMT) puts methyl groups on H3K9 (not shown). 2. HP1 binds to H3K9. 3. HP1 binds to histone methylase (HMT). 4. HMT methylates H3 in neighboring nucleosome, propagating the heterochromatin structure.

Which Chromatin Modifications are Associated with Repressed Heterochromatin & Epigenetic Silencing? 2

1. MethylationofH3K9 (lysine(K) at position 9 of the N-terminal tail of histone H3) 2. Methylation at C5 of cytosines in CpG dinucleotides

Repair Pathways

1. Mismatch Repair-coupled to DNA replication; repairs mismatches created during DNA replication 2. Base Excision Repair-removes damaged base and replaces it with a goodnucleotide. 3. Nucleotide Excision Repair-detects bulky damage and removes oligonucleotide harboring the damage. Recognize, Remove, Replacement*^^ 4. Strand break repair-detects single-strand and double-strand breaks in DNA and stitches the strands back together.

About what fraction of the human genome is found in exons and introns combined?

20%

How many base pairs are there in the human genome?

3 x 109

illumina

A next-generation sequencing method that works by fragmenting DNA and sequencing those fragments many times over

Mutant:

An organism or cell carrying one or more mutations

5-methylcytosine in CpG sequences

DNA methylation is established by DNA methyl transferase (DNMT), which puts a methyl group at the 5 position of a cytosine if it is followed by a guanine (CpG sequence).

Cooperative Interactions

Enhanceosome

whole-genome shotgun sequencing

Method of sequencing a genome in which sequenced fragments are assembled into the correct sequence in contigs by using only the overlaps in sequence.

Peptide Bond Formation

N in the amino group at the A site attacks the tRNA-aminoacyl ester linkage at the P site

Prokaryotes & eukaryotes have related multi-subunit

Prokaryotes & eukaryotes have related multi-subunit RNA polymerase structures

Molecular Mimicry plays a role in translational termination

RF structurally resemble tRNA

The protein component of a genome editing system that cleaves DNA

cas9

The 16S rRNA gene acts as a 'molecular fingerprint' that can be used to identify

different bacteria

The DNA Damage Response in human cells is complex. Mutations in many of the genes that code for DDR proteins are associated with

elevated rates of cancer!

Integration of transcription and RNA processing

factors are recruited to the phosphorylated CTD. These facilitate splicing and passage of polymerase through downstream chromatin. § A "stall factor" stops PolII shortly downstream of the initiation site. § Cap addition complex ("capping enzymes") binds to the Ser5-phosphorylated CTD and adds the cap to the 5' end of the mRNA.

Shh combinatorial control case study: Different cell types have same DNA but different proteins

for ex: cell type 1 : posterior nerves, cell type 2: anterior nerves same DNA -- express different protein activators -- GTFs bind DNA, which loops accordingly -- In both of these cases, Shh transcription is active, but the molecules that enable transcription are different.

Mutations are rare .....Why?

frequency of the 5 --> 3 polymerase activity of a typical DNA polymerase is ~10^-5 • Many DNA polymerases contain a proof-reading 3-->5 exonuclease activity that reduces the error frequency an additional 100-1000 fold by removing wrongly incorporated nucleotides • Post-replicative DNA repair --Of errors in replication --Of damaged DNA

decreasing or abolishing gene expression then assessing the outcome on a cell/organism

functional genomics

Proteins which are required for transcription of every gene in eukaryotes

general transcription factors

Lac repressor and many other bacterial DNA binding proteins bind to DNA using

helix- turn-helix DNA binding domains Lac Repressor binds to Operator as a dimer R-groups on one side of helix make contacts with major groove atoms

Contains genes which are rarely or never transcribed

heterochromatin

Heterochromatin

highly condensed chromatin Long-range repressed chromatin is called heterochromatin. • It is found in a constitutive (always present) form at telomeres and sequences that flank centromeres in many organisms, and in a developmentally regulated form in multi-cellular organisms. • Heterochromatin establishment requires the participation of specific non-histone chromosomal proteins that associate with the silenced DNA.

Nucleosome remodeling and histone acetylation are steps

in gene activation.

Tropomyosin

involved in muscle contraction.

Draw a simple drawing to show what this electron micrograph of 10nm particles represents. What kind of interaction(s) between the two main components of the structure shown hold them in this confirmation?

ionic interactions between the positively charged lysines in histones and the negative charges of DNA and (2) hydrogen bonds between histones and specific bases in the minor groove

Pre-Initiation Complex (PIC) is required for ____ transcription ____

is required for basal transcription in a test tube. Mediator recruited, General transcription factors released, and RNA polymerase recruited. Occurs at promoter

Protein that prevents transcription of structural genes of lac operon

lac repressor

Compounds that induces expression of lac operon

lactose, IPTG

Amino acid that is commonly found in histones

lysine

Types of Prokaryotic RNA • __________________ • Encode proteins • ___________________ • Bring correct amino acid to mRNA • ____________________ • Major component of ribosomes 2 &3= "Functional" or "Structural" RNAs

messenger RNA (mRNA) - encode proteins transfer RNA (tRNA) - Bring correct amino acid to mRNA ribosomal RNA (rRNA) - Major component of ribosomes • Each made by transcription of DNA using the same RNA polymerase

Lysine on histone tails can be methylated. Lysine methylation has varying effects on transcription. for this class, know that methylation of H3K9 marks

methylation of H3K9 marks *repressed* chromatin

How does the function of miRNA (microRNA) differ when it is an exact base pair match to its target versus when it is just a close match to the target? In your response, please identify which type of molecule is targeted by an miRNA.

miRNA binding to a target mRNA sequence causes degradation when it is an exact match, but just temporarily stalls translation (or decreases amount of translation) when it is a close match

Repressor binding site (e.g in lacoperon)

operator

Cluster of genes regulated by a single promoter

operon

cap addition occurs shortly after transcription initiation. It is coordinated by the

phosphorylated RNA pol II CTD (c terminal domain)

Generated by untemplated nucleotide polymerization

ploy A tail

Compare & contrast gene expression in prokaryotic and eukaryotic cells

prokaryote • No nucleus • Circular chromosome • DNA replication, trx and trnsl in one compartment • A lot going on at once eukaryote • Nucleus Linear • DNA replication and trx in nucleus • RNAs processed à cytoplasm • Translation in cytoplasm • Trx and translation uncoupled

Eukaryotic Cell Cycle Phases - G1, S, G2, & M Passage through the Restriction Point requires growth factor signaling, which activates a

protein kinase

Targets of activation domains are recruited to the promoter by ___ among these targets are co-activators

protein-protein interactions

Allosteric effectors (= inducers) bind to

regulatory proteins, influence DNA binding

Gene expression is controlled by the binding of regulatory proteins to Negative Regulation: Positive Regulation

regulatory regions of genes or operons •Negative Regulation: Repressor proteins bind to DNA, prevents RNA polymerase from binding and/or initiating transcription •In absence of Repressor: transcription is initiated Positive Regulation: Activator proteins help recruit RNA polymerase to promoter, activate transcription • If no activator present: no transcription (or only low, basal level)

Protein that terminates RNA-synthesis

rho protein

RNA polymerase subunit that recognizes promoter

sigma

Cas9 nuclease targeted to desired DNA sequence by a ____________________encoded by repeat sequences in bacterial CRISPR locus

single guide RNA (sgRNA)

Features of the Genetic Code: Punctuation:

start codon (usually AUG [methyl]) and stop codons An Open Reading Frame (ORF) is a long string of amino acid-specifying codons that begins with an AUG codon and is not interrupted by stop codons. Codons in reading frame are non-overlapping • 3 different reading frames on each strand, 6 reading frames in total

The N-terminal tails of histones are targets of

targets of extensive modification

Many human genes have multiple enhancers, spread over large distances, each of which is responsible for activating expression of the gene at a specific time and/or place. An example is

the Sonic hedgehog (Shh)gene, a developmental regulator, that has enhancers spread over more than a megabase of DNA! Shh mutants have larva that look like hedgehogs.

"Indel" mutations are

the insertion or deletion of a small number of base pairs, due to slippage during replication

Self-splicing Introns

the intron RNA folds into a structure that catalyzes its own removal. The RNA functions as an enzyme or a "ribozyme".

mRNA processing occurs in the

the nucleus, prior to export to the cytoplasm

RNA Polymerase transcribes from

the template strand.

CRISPR/Cas9 • Fundamental understanding of how this system works in bacteria has resulted in development of methods to produce _______________________ • _____________________________________ • Nuclease activity is targeted to desired DNA sequence by an ___________: that is easily generated by chemical synthesis of appropriate DNA template.

to produce sequence sequence specific breaks in double stranded (ds) DNA in eukaryotic cells targeted to desired DNA sequence by an * guide rna*

Chromatin remodelers use the energy of ATP hydrolysis

to reposition or restructure nucleosomes

RNA synthesis

transcription

Think of chromatin as a compact structure that needs be that needs to be made accessible for

transcription, replication, and DNA repair.

Problem: How do you encode 20 amino acids with only 4 different letters?

triplet code 4^3

Histone lysine acetylation weakens

weakens DNA binding - DNA is *acidic* because of negatively charged phosphates. - Histones are *basic* because of abundant positively charged lysine and arginine amino acid residues. - Lysines in amino terminal tails can be modified by acetylation, which neutralizes the positive charge.

Initiation of Translation in Prokaryotes

• 30S and 50S subunits not yet associated. Kept separated by Initiation Factor 3 - IF3 • Initiation Factor 2 - IF2: delivers a special tRNA = initiator tRNAf charged with formylmethionine (fMet) to the P site (usually AUG) • Establishes ORF • IF2 ensures that only initiator tRNA-fMet enters P site • Once tRNA-fMet in place, 50S su of ribosome can bind • IF2 is a type of G-protein: Switches between GTP- and GDP-bound forms due to GTPase activity of the protein • When 50S subunit binds to 30S, GTP is hydrolyzed to GPD & IF2 released. • Ribosome is properly positioned, ready to elongate

A Mammalian RNA Pol II gene with combinatorial control: A case study - genes - +/- of inducer

• A gene named Sonic hedgehog(Shh) is a signal that is used only at specific times/places during development. • It has multiple"modular" enhancers(blue, red, green and yellow ), each responsible for activating expression at a specific time and place. Each enhancer must bind a particular combination of transcription factors in order to be active. • Mediator "mediate" interactions between Enhancer- Bound factors and Promoter-Bound Factors

Combinations of Cooperatively-Bound Transcription Activators Regulate Eukaryotic Genes:

• Activation requires multiple factors, some of which bind to DNA cooperatively. • Different genes require different combinations of activators. • Activation of a the same gene at different times and places also involves different combinations of activators

Analysis of gut microbiome composition in ob/ob and normal mice Do the microbiota in the obese mouse impact the mouse phenotype?

• All mice were fed the same carbohydrate-rich diet • Observation: ob/ob mice have altered microbiome composition Result: The microbes from ob/ob mice transmit an increase in adiposity to lean germ-free recipients...what about humans?

The Human Microbiome

• As soon as baby is born she is populated by microbes • Colonization occurs where the body meets the external environment: - Examples: Skin, Mouth, nose, Gastrointenstinal (GI) tract • Most internal tissues are believed to be sterile (unless there is an infection) • "Microbiota often referred to as"commensals"-only microbe benefits - old view. • New view: mutualistic interaction -both host and microbe benefit

Positive control Activation of the lac operon in the presence of lactose (inducer) but low glucose

• Cell does not want to activate expression of lac operon if there is plenty of Glucose around • Glucose is a better carbon source. • What do you think cell might use as an "inducer" to convert CAP into an active DNA binding protein? • An indicator of energy • status of cell. - cAMP

A typical eukaryotic protein coding gene

• Coding sequence: exons & introns • One ORF per trx (no operons) • 5'and 3' untranslated regions (UTR) (poly A signal) • Promoter & upstream regulatory regions • Transcription start site (+1) • Trx termination

The major groove is not only accessible to DNA binding proteins but is also the most information-rich

• DNA binding proteins can make contact with different H-bond donors and acceptors • Allows for "recognition" of specific binding sites

Gene expression important in:

• Development • Environmental response

CRISPR/Cas9 & Genome Editing

• Efficient & precise tool for genome editing • Used to address fundamental research questions in many organisms • Gene therapy in mammals • Introducing improved traits into plants

Genomics: The study of the structure and function of whole genomes

• Genome: The complete set of genes or genetic material present in a cell or organism. • Genome analysis involves: 1. Sequencing entire genome to determine its DNA sequence 2. Mapping genes to specific locations on chromosome. 3. Examine gene expression patterns 4. Determining the function of genes: • Reverse genetics • Proteomics 5. Investigating interactions between genes

germ-free (gnotobiotic) animal models

• Gnotobiotic ("known life") animals are reared germ- free in sterile isolators. • Germ-free animals intentionally colonized later in life with microbial communities researchers want to study. • Gnotobiotic animal models allow researchers to: - build microbial communities from scratch in vivo - investigate how the addition/removal of particular microbes impacts community biology - test the influence of different microbial communities on the host.

Elongation Phase

• If new tRNA in A site is a match, accepted • Conformational change of A site • EF-Tu-GTP hydrolyzed, leaves • AA end of tRNAs in P and A sites in close proximity: • Peptide bond formed: • f-MET transferred to AA on tRNA in A site (peptide bond) • EF-G appears, fits into A site, shifts tRNA into new sites: • Ribosome moves forward

Hereditary Nonpolyposis Colon Cancer (HNPCC Genetic / Biochemical defect:

• Increased incidence of colon cancer, as well as some non- colonic cancers • Common; associated with ~1-5% of colon cancer cases • Caused by mutations in ~6 genes encoding human mismatch repair enzymes, particularly the human versions of MutL and MutS (MLH1 & MSH2) • 100-1000 x increase in mutation rate in HNPCC cells • Estimated that up to 1/200 individuals in Western populations carry such a mutation; these individuals have ~80% chance of developing colon cancer

An aminoacyl-tRNA is sometimes called a charged tRNA

• Intermediates in protein synthesis are amino acid esters. • Not very stable. • Protected by association with EF-Tu translation factor!

Homology Directed Repair (HDR)

• Involves *homologous recombination*: Exchange of genetic information between DNA molecules that have high levels of homology (sequence identity) • Occurs frequently in bacteria, yeast. Also occurs in mice, other mammals. • Has been used to "knockout" every predicted ORF in yeast. • Many mouse genes also have been knocked out by this method.

Do gut microbiota play a critical role in obtaining calories ?

• Mice lacking gut microbes do not obtain as many calories from their food (are not as fat) as mice that have normal gut microbes • Mice without gut microbes actually eat more than those with gut microbes (are they more hungry?) • Conclude: Mice with an intact gut microbiome may be more efficient at harvesting energy from food. yes

7 Basic structure of a typical bacterial protein- coding gene

• Open Reading Frame (ORF) = Protein coding sequence • Start codon: ATG • Stop codon: TAG, TGA, TAA • Transcription Start Site (TSS) • Promoter: RNA polymerase binds here, determines trx start -10, - 35 regions • Transcription terminator • 5' and 3' untranslated regions: Regulation

Basic structure of a typical bacterial protein- coding gene

• Open Reading Frame (ORF) = Protein coding sequence • Start codon: ATG • Stop codon: TAG, TGA, TAA • Transcription Start Site (TSS) • Promoter: RNA polymerase binds here, determines trx start -10, - 35 regions • Transcription terminator • 5' and 3' untranslated regions: Regulation

Catabolite Repression via CAP

• Positive regulation of catabolic operons • Extra layer of regulation ensures that bacteria does not invest energy in utilizing other sugars until glucose (easily digested) is exhausted. • Glucose is a catabolite of lactose

A typical prokaryotic operon

• Prokaryotic genes often organized in operons = multiple ORFs present in one transcript - each has own ATG, stop • Transcription starts at specific site = +1 • Determined by promoter • Transcription termination specified • No introns: 1o trx = mRNA • This is the lac operon

Culture-independent methods for describing microbial communities

• Rely on sequencing DNA of the organisms within a community • Metagenomics: the study of genetic material recovered directly from environmental samples • Two approaches: 1. 'Targeted' metagenomics: sequence single genetic locus (e.g., 16S rRNA gene) within community DNA 2. 'Shotgun' metagenomics: sequence community DNA in unbiased manner (i.e., whole genomes) • Allows analysis of various functions in community

Termination of Transcription: 2 ) Rho-dependent mechanism

• Rho (r) protein binds to rut site in 3' end of the RNA • Acts as a helicase to disrupt the RNA-DNA hybrid of a paused polymerase • Facilitates dissociation of RNA from DNA template

Elongation of Translation

• The Elongation Factor EF-Tu, another G-protein, complexes in its GTP-bound form with a charged tRNA = tRNA-AA • A "ternary complex" • tRNA-AA loaded into A site of the ribosome • EF-Tu is released upon GTP hydrolysis and recycled

Features of the Genetic Code:

• Triplet code: 64 different 3 nucleotide "codons"; nonoverlapping; 3 different "reading frames" on each strand; 6 possible reading frames; redundancy of genetic code; only 20 amino acids to be encoded by 64 possible codons in the triplet code; most amino acids specified by two or more codons; synonymous codons specify same amino acid --- punctuation

Partial diploids allowed Jacob and Monod to test whether regulatory elements of the lac operon worked in cis or trans.

• Useofpartialdiploidsa common genetic approach. - Extra DNA fragment carried on a plasmid (F') - Two copies of part of the genome (a.k.a. partial diploid) Use of partial diploids allows geneticists to ask if second copy of DNA on a plasmid can restore wild- type phenotype to a mutant. - Determine dominant/recessive relationship of alleles (genetic complementation). - If a genetic element must be physically adjacent to the lac operon to be functional, it functions in "cis". - If a genetic element can be located on a separate piece of DNA, it it functions in "trans".

Mismatch Repair in E. coli

• coupled to replication • MutS protein recognizes the mismatch (or small deletion). • Recruits MutL and MutH • Single-stranded cleavage near the mismatch (at a GATC sequence) on the newly replicated strand • Exonuclease removes mismatch and surrounding nucleotides • DNA pol + DNA ligase fill in the gap

The phosphorylated CTD provides a binding site for factors involved in both:

• processing the transcript, and • regulating the "activity" of transcribed chromatin. Required to "open" chromatin to allow the passage of RNA Polymerase, then to close it after the polymerase has passed through.

Elongation

• s dissociates after initiation • RNA polymerase core enzyme carries out synthesis • Transcription bubble: ~17 bp unwound • RNA-DNA hybrid: ~ 8-10 bp • Trx Rate: 50 nucleotides per second • Error rate 10^-4 - 10^-5